1,2,4-Oxadiazole Derivatives as Immunomodulators

ABSTRACT

The present invention relates to pharmaceutical compositions of 1,2,4-oxadiazole compounds or a pharmaceutically acceptable salt thereof of formula (I) 
     
       
         
         
             
             
         
       
     
     In the formula Q is O, R 1  is the side chain of Ser, R 2  is —CO-Thr, R 3  is the side chain of Asn or Glu, and R 4 , R 5  and R 6  are each H.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. § 120 of pendingapplication U.S. Ser. No. 17/192,279, filed Mar. 4, 2021, which is acontinuation under 35 U.S.C. § 120 of U.S. Ser. No. 16/806,872, filedMar. 2, 2020, now U.S. Pat. No. 10,961,205, which is a continuationunder 35 U.S.C. § 120 of U.S. Ser. No. 16/192,030, filed Nov. 15, 2018,now U.S. Pat. No. 10,590,093, which is a continuation under 35 U.S.C. §120 of application U.S. Ser. No. 15/713,671, filed Sep. 24, 2017, nowU.S. Pat. No. 10,173,989, which is a continuation under 35 U.S.C. § 120of U.S. Ser. No. 15/298,539, filed Oct. 20, 2016, now U.S. Pat. No.9,771,338, which is a continuation under 35 U.S.C. § 120 of applicationU.S. Ser. No. 14/478,759, filed Sep. 5, 2014, now abandoned, whichclaims benefit of priority of Indian provisional application number4011/CHE/2013, filed on Sep. 6, 2013, now abandoned, and for which acertified copy thereof is found in the file wrapper of U.S. Ser. No.15/298,539, the entirety of all of which are hereby incorporated byreference.

BACKGROUND OF THE INVENTION Technical Field of the Invention

The present invention relates to 1,2,4-oxadiazole and 1,2,4-thiadiazolecompounds and their derivatives therapeutically useful as immunemodulators. The invention also relates to pharmaceutical compositionscomprising the said 1,2,4-oxadiazole and 1,2,4-thiadiazole compounds andtheir derivatives as therapeutic agents.

Description of the Related Art

Programmed cell death-1 (PD-1) is a member of the CD28 superfamily thatdelivers negative signals upon interaction with its two ligands, PD-L1or PD-L2. PD-1 and its ligands are broadly expressed and exert a widerrange of immunoregulatory roles in T cells activation and tolerancecompared with other CD28 members. PD-1 and its ligands are involved inattenuating infectious immunity and tumor immunity, and facilitatingchronic infection and tumor progression. The biological significance ofPD-1 and its ligand suggests the therapeutic potential of manipulationof PD-1 pathway against various human diseases (Ariel Pedoeem et al.,Curr Top Microbiol Immunol. (2011); 350:17-37).

T-cell activation and dysfunction relies on direct and modulatedreceptors. Based on their functional outcome, co-signaling molecules canbe divided as co-stimulators and co-inhibitors, which positively andnegatively control the priming, growth, differentiation and functionalmaturation of a T-cell response (Li Shi, et al., Journal of Hematology &Oncology 2013, 6:74).

Therapeutic antibodies that block the programmed cell death protein-1(PD-1) immune checkpoint pathway prevent T-cell down regulation andpromote immune responses against cancer. Several PD-1 pathway inhibitorshave shown robust activity in various phases of on-going clinical trials(RD Harvey, Clinical Pharmacology & Therapeutics (2014); 96 2, 214-223).

Programmed death-1 (PD-1) is a co-receptor that is expressedpredominantly by T cells. The binding of PD-1 to its ligands, PD-L1 orPD-L2, is vital for the physiological regulation of the immune system. Amajor functional role of the PD-1 signaling pathway is the inhibition ofself-reactive T cells, which serve to protect against autoimmunediseases. Elimination of the PD-1 pathway can therefore result in thebreakdown of immune tolerance that can ultimately lead to thedevelopment of pathogenic autoimmunity. Conversely, tumor cells can attimes co-opt the PD-1 pathway to escape from immunosurveillancemechanisms. Therefore, blockade of the PD-1 pathway has become anattractive target in cancer therapy. Current approaches include sixagents that are either PD-1 and PD-L1 targeted neutralizing antibodiesor fusion proteins. More than forty clinical trials are underway tobetter define the role of PD-1 blockade in variety of tumor types(Hyun-Tak Jin et al., Clinical Immunology (Amsterdam, Netherlands)(2014), 153(1), 145-152).

International applications WO 01/14557, WO 02/079499, WO 2002/086083, WO03/042402, WO 2004/004771, WO 2004/056875, WO2006121168, WO2008156712,WO2010077634, WO2011066389, WO2014055897, WO2014059173, WO2014100079 andUS patent U.S. Ser. No. 08/735,553 report PD-1 or PD-L1 inhibitoryantibodies or fusion proteins.

Further, International applications, WO2011161699, WO2012/168944,WO2013144704 and WO2013132317 report peptides or peptidomimeticcompounds which are capable of suppressing and/or inhibiting theprogrammed cell death 1 (PD1) signaling pathway.

Still there is a need for more potent, better and/or selective immunemodulators of PD-1 pathway. The present invention provides1,2,4-oxadiazole and 1,2,4-thiadiazole compounds which are capable ofsuppressing and/or inhibiting the programmed cell death 1 (PD1)signaling pathway.

SUMMARY OF THE INVENTION

In accordance with the present invention, 1,2,4-oxadiazole and1,2,4-thiadiazole compounds or a pharmaceutically acceptable salt or astereoisomer thereof, provided which are capable of suppressing and/orinhibiting the programmed cell death 1 (PD1) signaling pathway.

In one aspect, the present invention provides 1,2,4-oxadiazole and1,2,4-thiadiazole compounds of formula (I):

wherein,

Q is S or O;

R₁ is a side chain of amino acid Ser or Thr, optionally substituted withalkyl or acyl;

R₂ is hydrogen or —CO-Aaa;

Aaa is an amino acid residue Thr or Ser; wherein a C-terminus thereof isa free terminus, is amidated or is esterified;

R₃ is a side chain of amino acid Asn, Asp, Gln or Glu;

is an optional bond;

R₄ and R₅ independently are hydrogen or absent;

R₆ is hydrogen, alkyl or acyl;

or a pharmaceutically acceptable salt or a stereoisomer thereof.

In a further aspect of the present invention, there is provided apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt or a stereoisomer and processes forpreparing thereof.

In yet another aspect of the present invention, there is providedmethods for suppressing and/or inhibiting the programmed cell death 1(PD1) signaling pathway in a subject by administering 1,2,4-oxadiazoleand 1,2,4-thiadiazole compounds of formula (I) or a pharmaceuticallyacceptable salt or a stereoisomer thereof or pharmaceutical compositionsthereof.

In yet another aspect of the present invention, there is providedmethods for inhibiting growth of tumour cells and/or metastasis in asubject by administering 1,2,4-oxadiazole and 1,2,4-thiadiazolecompounds of formula (I) or a pharmaceutically acceptable salt or astereoisomer thereof or pharmaceutical compositions thereof.

In yet another aspect of the present invention, there is providedmethods for treating an infectious disease or a bacterial, viral andfungal infections in a subject by administering 1,2,4-oxadiazole and1,2,4-thiadiazole compounds of formula (I) or a pharmaceuticallyacceptable salt or a stereoisomer thereof or pharmaceutical compositionsthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsand certain embodiments of the invention briefly summarized above areillustrated in the appended drawings. These drawings form a part of thespecification. It is to be noted, however, that the appended drawingsillustrate preferred embodiments of the invention and therefore are notto be considered limiting in their scope.

FIGS. 1A-1B depict the chemical synthetic scheme for Compound 1.

FIG. 2 depicts the chemical synthetic scheme for Compound 2.

FIG. 3 depicts the chemical synthetic scheme for Compound 7.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides 1,2,4-oxadiazole and 1,2,4-thiadiazolecompounds as therapeutic agents useful in methods for treating disordersvia immunopotentiation comprising inhibition of immunosuppressive signalinduced due to PD-1, PD-L1, or PD-L2 and therapies using them.

Each embodiment is provided by way of explanation of the invention, andnot by way of limitation of the invention. In fact, it will be apparentto those skilled in the art that various modification and variations canbe made in the present invention without departing from the scope orspirit of the invention. For instance, features illustrated or describedas part of one embodiment can be used on another embodiment to yield astill further embodiment. Thus it is intended that the present inventioncover such modifications and variations as come within the scope of theappended claims and their equivalents. Other objects, features, andaspects of the present invention are disclosed in, or are obvious from,the following detailed description. It is to be understood by one ofordinary skill in the art that the present discussion is a descriptionof exemplary embodiments only, and is not to be construed as limitingthe broader aspects of the present invention.

In one embodiment, the present invention relates to compounds of formula(I)

wherein,

Q is S or O;

R₁ is a side chain of amino acid Ser or Thr, optionally substituted withalkyl or acyl;

R₂ is hydrogen or —CO-Aaa;

Aaa is an amino acid residue Thr or Ser; a C-terminus thereof is a freeterminus, is amidated or is esterified;

R₃ is side chain of amino acid Asn, Asp, Gln, or Glu;

is an optional bond;

R₄ and R₅ independently are hydrogen or absent;

R₆ is hydrogen, alkyl or acyl;

or a pharmaceutically acceptable salt or a stereoisomer thereof.

In another embodiment, the present invention provides compounds offormula (IA)

or a pharmaceutically acceptable salt or a stereoisomer thereof;wherein,

R₁, R₃ and Aaa are as defined in formula (I).

In yet another further embodiment, the present invention providescompounds of formula (IB)

or a pharmaceutically acceptable salt or a stereoisomer thereof;wherein,

R₁ and R₃ are the same as defined in formula (I).

In yet another further embodiment, for the compound according to formula(I) Q is S.

In yet another further embodiment, for the compound according to formula(I) R₄ is hydrogen.

In yet another further embodiment, for the compound according to formula(I) R₅ is hydrogen.

The embodiments below are illustrative of the present invention and arenot intended to limit the claims to the specific embodimentsexemplified.

According to one embodiment, specifically provided are compounds of theformula (I), (IA) and (IB) in which R₁ is a side chain of Ser.

According to another embodiment, specifically provided are compounds ofthe formula (I), (IA) and (IB) in which R₁ is a side chain of Thr.

According to yet another embodiment, specifically provided are compoundsof the formula (I) in which R₂ is hydrogen.

According to yet another embodiment, specifically provided are compoundsof the formula (I) in which R₂ is —CO-Aaa.

According to yet another embodiment, specifically provided are compoundsof the formula (I) and (IA) in which Aaa is Thr.

According to yet another embodiment, specifically provided are compoundsof the formula (I) and (IA) in which Aaa is Ser.

According to yet another embodiment, specifically provided are compoundsof the formula (I), (IA) and (IB) in which R₃ is a side chain of Asn.

According to yet another embodiment, specifically provided are compoundsof the formula (I) and (IA) in which R₃ is a side chain of Asp.

According to yet another embodiment, specifically provided are compoundsof the formula (I) and (IA) in which R₃ is a side chain of Gln.

According to yet another embodiment, specifically provided are compoundsof the formula (I) and (IA) in which R₃ is side a chain of Glu.

According to yet another embodiment, specifically provided are compoundsof the formula (I) in which R₁ is a side chain of Ser or Thr; R₂ is—CO-Aaa; Aaa is an amino acid residue Thr or Ser; wherein C-terminus isfree; and R₃ is a side chain of Asn or Glu.

According to yet another embodiment, specifically provided are compoundsof the formula (I) in which Q is O.

According to yet another embodiment, specifically provided are compoundsof the formula (I) in which R₁ is a side chain of Ser, optionallysubstituted with C₁₋₅ alkyl such as methyl.

According to yet another embodiment, specifically provided are compoundsof the formula (I) in which R₆ is hydrogen.

According to yet another embodiment, specifically provided are compoundsof the formula (I) in which R₆ is acyl such as butyryl.

According to yet another embodiment, specifically provided are compoundsof the formula (I) in which R₄ and R₅ are absent.

According to yet another embodiment, specifically provided are compoundsof the formula (I) in which C-terminus of Aaa is free (e.g. —CO₂H form).

According to yet another embodiment, specifically provided are compoundsof the formula (I) in which C-terminus of Aaa is esterified (e.g. —CO₂Meform).

According to yet another embodiment, specifically provided are compoundsof the formula (I) in which C-terminus of Aaa is amidated (e.g. —CONH₂form).

According to yet another embodiment, specifically provided are compoundsof the formula (I), (IA) and (IB) in which one, more or all amino acid/sis/are D amino acid/s.

In an embodiment, specific compounds of formula (I) without anylimitation are enumerated in Table 1 or a pharmaceutically acceptablesalt or a stereoisomer thereof.

TABLE 1 Compound No. Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising the compound as disclosed, and a pharmaceuticallyacceptable carrier or diluent.

In another embodiment, the pharmaceutical composition further comprisingat least one of an anticancer agent, chemotherapy agent, orantiproliferative compound.

The compounds as disclosed in the present invention are formulated forpharmaceutical administration.

In one embodiment, the present invention provides use of the compoundsas disclosed in the present invention for the preparation of amedicament.

In another embodiment, the present invention provides use of thecompounds as disclosed in the present invention for the preparation of amedicament for the treatment of cancer or infectious disease.

In one embodiment, the present invention provides use of the compoundsas disclosed in the present invention for the preparation of amedicament for the treatment of bacterial, viral and fungal infections.

In one embodiment, the present invention provides a method of treatmentof cancer, wherein the method comprises administration of an effectiveamount of the compound of the present invention or of a pharmaceuticalcomposition thereof to the subject in need thereof.

In another embodiment the present invention provides a method ofmodulating an immune response mediated by PD-1 signaling pathway in asubject, comprising administering to the subject a therapeuticallyeffective amount of the compound of the present invention or apharmaceutical composition thereof such that the immune response in thesubject is modulated.

In yet another embodiment the present invention provides a method ofinhibiting growth of tumour cells and/or metastasis in a subject,comprising administering to the subject a therapeutically effectiveamount of compound of the present invention or a pharmaceuticalcomposition thereof capable of inhibiting the programmed cell death 1(PD1) signaling pathway.

The said tumour cells include cancer such as, but not limited to, bonecancer, cancer of the head or neck, pancreatic cancer, skin cancer,cutaneous or intraocular malignant endometrium, carcinoma of the cervix,carcinoma of the vagina, carcinoma of the vulva, Hogkin's Disease,non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, cancer of the thyroid gland,cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma ofsoft tissue, cancer of the urethra, cancer of the penis, chronic oracute leukemias including acute myeloid leukemia, chronic myeloidleukemia acute lymphoblastic leukemia, chronic lymphocytic leukemia,solid tumours of childhood, lymphocytic lymphoma cancer of the bladder,cancer of the kidney or reter, carcinoma of the renal pelvis, neoplasmof the central nervous system (CNS), primary CNS lymphoma, tumourangiogenesis, spinal axis tumour, brain stem glioma, pituitary adenoma,Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-celllymphoma, environmentally induced cancers including those induced byasbestos, and combinations of said cancers.

In yet another further embodiment the present invention provides amethod of treating an infectious disease in a subject comprisingadministering to the subject a therapeutically effective amount of thecompound of the present invention or a pharmaceutical compositionthereof capable of inhibiting the programmed cell death 1 (PD1)signalling pathway such that the subject is treated for the infectiousdisease.

Still yet another embodiment of the present invention provides a methodof treating bacterial, viral and fungal infections in a subjectcomprising administering to the subject a therapeutically effectiveamount of the compound of the present invention or a pharmaceuticalcomposition thereof capable of inhibiting the programmed cell death 1(PD1) signaling pathway such that the subject is treated for thebacterial, viral and fungal infections.

The infectious disease includes but not limited to HIV, Influenza,Herpes, Giardia, Malaria, Leishmania, the pathogenic infection by thevirus Hepatitis (A, B, & C), herpes virus (e.g., VZV, HSV-I, HAV-6,HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus,flaviviruses, echovirus, rhinovirus, coxsackie virus, cornovirus,respiratory syncytial virus, mumps virus, rotavirus, measles virus,rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus,papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus andarboviral encephalitis virus, pathogenic infection by the bacteriachlamydia, rickettsial bacteria, mycobacteria, staphylococci,streptococci, pneumonococci, meningococci and conococci, klebsiella,proteus, serratia, pseudomonas, E. coli, legionella, diphtheria,salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague,leptospirosis, and Lyme's disease bacteria, pathogenic infection by thefungi Candida (albicans, krusei, glabrata, tropicalis, etc.),Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), GenusMucorales (mucor, absidia, rhizophus), Sporothrix schenkii, Blastomycesdermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis andHistoplasma capsulatum, and pathogenic infection by the parasitesEntamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoebasp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii,Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosomacruzi, Leishmania donovani, Toxoplasma gondi, Nippostrongylusbrasiliensis.

The compounds of the present invention may be used as single drugs or asa pharmaceutical composition in which the compound is mixed with variouspharmacologically acceptable materials.

The pharmaceutical composition is usually administered by oral orinhalation routes, but can be administered by parenteral administrationroute. In the practice of this invention, compositions can beadministered, for example, by orally, intravenous infusion, topically,intraperitoneally, intravesically or intrathecally. Examples of theparenteral administration includes but not limited to intraarticular (inthe joints), intravenous, intramuscular, intradermal, intraperitoneal,and subcutaneous routes, include aqueous and non-aqueous, isotonicsterile injection solutions, which can contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, and aqueous and non-aqueous sterilesuspensions that can include suspending agents, solubilizers, thickeningagents, stabilizers, and preservatives. Oral administration, parenteraladministration, subcutaneous administration and intravenousadministration are the preferred methods of administration.

The dosage of the compounds of the present invention varies depending onage, weight, symptom, therapeutic efficacy, dosing regimen and/ortreatment time. Generally, they may be administered by oral orinhalation routes, in an amount of 1 mg to 100 mg per time, from once acouple of days, once 3 days, once 2 days, once a day to a couple oftimes a day, in the case of an adult, or continuously administered byoral or inhalation routes from 1 to 24 hours a day. Since the dosage isaffected by various conditions, an amount less than the above dosage maysometimes work well enough, or higher dosage may be required in somecases.

The compounds of the present invention may be administered incombination with other drugs for (1) complementation and/or enhancementof prevention and/or therapeutic efficacy of the preventive and/ortherapeutic drug of the present invention, (2) dynamics, absorptionimprovement, dosage reduction of the preventive and/or therapeutic drugof the present invention, and/or (3) reduction of the side effects ofthe preventive and/or therapeutic drug of the present invention.

A concomitant medicine comprising the compounds of the present inventionand other drug may be administered as a combination preparation in whichboth components are contained in a single formulation, or administeredas separate formulations. The administration by separate formulationsincludes simultaneous administration and administration with some timeintervals. In the case of the administration with some time intervals,the compound of the present invention can be administered first,followed by another drug or another drug can be administered first,followed by the compound of the present invention. The administrationmethod of the respective drugs may be the same or different.

The dosage of the other drug can be properly selected, based on a dosagethat has been clinically used. The compounding ratio of the compound ofthe present invention and the other drug can be properly selectedaccording to age and weight of a subject to be administered,administration method, administration time, disorder to be treated,symptom and combination thereof. For example, the other drug may be usedin an amount of 0.01 to 100 parts by mass, based on 1 part by mass ofthe compound of the present invention. The other drug may be acombination of two or more kind of arbitrary drugs in a properproportion. The other drug that complements and/or enhances thepreventive and/or therapeutic efficacy of the compound of the presentinvention includes not only those that have already been discovered, butthose that will be discovered in future, based on the above mechanism.

Diseases on which this concomitant use exerts a preventive and/ortherapeutic effect are not particularly limited. The concomitantmedicine can be used for any diseases, as long as it complements and/orenhances the preventive and/or therapeutic efficacy of the compound ofthe present invention.

The compound of the present invention can be used with an existingchemotherapeutic concomitantly or in a mixture form. Examples of thechemotherapeutic include an alkylation agent, nitrosourea agent,antimetabolite, anticancer antibiotics, vegetable-origin alkaloid,topoisomerase inhibitor, hormone drug, hormone antagonist, aromataseinhibitor, P-glycoprotein inhibitor, platinum complex derivative, otherimmunotherapeutic drugs and other anticancer drugs. Further, it can beused with a cancer treatment adjunct, such as a leucopenia (neutropenia)treatment drug, thrombocytopenia treatment drug, antiemetic and cancerpain intervention drug, concomitantly or in a mixture form.

In one embodiment, the compound(s) of the present invention can be usedwith other immunomodulators and/or a potentiating agent concomitantly orin a mixture form. Examples of the immunomodulator include variouscytokines, vaccines and adjuvants. Examples of these cytokines, vaccinesand adjuvants that stimulates immune responses include but not limitedto GM-CSF, M-CSF, G-CSF, interferon-α, β, or γ, IL-1, IL-2, IL-3, IL-12,Poly (I:C) and C_(p)G.

In another embodiment, the potentiating agents includes cyclophosphamideand analogs of cyclophosphamide, anti-TGFβ and Imatinib (Gleevac), amitosis inhibitor, such as paclitaxel, Sunitinib (Sutent) or otherantiangiogenic agents, an aromatase inhibitor, such as letrozole, an A2aadenosine receptor (A2AR) antagonist, an angiogenesis inhibitor,anthracyclines, oxaliplatin, doxorubicin, TLR4 antagonists, and IL-18antagonists.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in artto which the subject matter herein belongs. As used herein, thefollowing definitions are supplied in order to facilitate theunderstanding of the present invention.

As used herein, the term ‘compound(s)’ refers to the compounds disclosedin the present invention.

As used herein, the term “comprise” or “comprising” is generally used inthe sense of include, that is to say permitting the presence of one ormore features or components.

As used herein, the term “including” as well as other forms, such as“include”, “includes,” and “included,” is not limiting.

As used herein, the term “optionally substituted” refers to thereplacement of one or more hydrogen radicals in a given structure withthe radical of a specified substituent including, but not limited to:alkyl, alkoxy, acyl, halo, and hydroxyl. It is understood that thesubstituent may be further substituted.

As used herein the term “alkyl” refers to a hydrocarbon chain radicalthat includes solely carbon and hydrogen atoms in the backbone,containing no unsaturation, having from one to twenty carbon atoms(i.e., C₁₋₂₀ alkyl) or one to ten carbon atoms (i.e., C₁₋₁₀ alkyl) orone to five carbon atoms (i.e., C₁₋₅ alkyl) and which is attached to therest of the molecule by a single bond, e.g., including but not limitedto methyl, ethyl, propyl, butyl, isobutyl, sec-butyl, tert-butyl,isopentyl or neopentyl. Unless set forth or recited to the contrary, allalkyl groups described or claimed herein may be straight chain orbranched, substituted or unsubstituted.

As used herein, the term “acyl” refers to RC(O)—, wherein R is alkyl asdefined above. Examples of acyl group include, but are not limited to—C(O)CH₃, —C(O)CH₂CH₃, —C(O)(CH₂)₂CH₃, —C(O)(CH₂)₃CH₃, —C(O)(CH₂)₄CH₃,—C(O)(CH₂)₅CH₃—C(O)(CH₂)₆CH₃ and —C(O)(CH₂)₈CH₃.

As used herein, the term “amidated C-terminus” refers to that theC-terminal of the amino acid in amide form.

As used herein, the term “amide form” refers to primary, secondaryand/or tertiary amides and may be represented by the formula—C(O)NR_(x)R_(y), wherein each of R_(x) and R_(y) independentlyrepresents hydrogen or alkyl.

As used herein, the term “amino” refers to —NH₂ group. Unless set forthor recited to the contrary, all amino groups described or claimed hereinmay be substituted or unsubstituted.

As used herein, the term “amino acid” refers to amino acids having L orD stereochemistry at the alpha carbon. Optional substituent on aminoacid means replacement of one or more hydrogen radicals in a givenstructure with the radical of a specified substituent, in case of aminoacid containing hydroxyl group such as Serine or Threonine, the hydroxylgroup can be substituted with the specified substituent.

As used herein, the term “aryl” refers to C₄-C₁₀ carbocyclic aromaticsystem containing one or two rings wherein such rings may be fused.Examples of aryl groups include, but are not limited to phenyl andnaphthyl.

As used herein, the term “arylalkyl” refers to an aryl group as definedabove directly bonded to an alkyl group (e.g., benzyl and the like).

As used herein, the term “carboxylic acid” refers to —COON group.

As used herein, the term “coupling agent” means a compound that reactswith the hydroxyl moiety of a carboxy moiety thereby rendering itsusceptible to nucleophilic attack. Coupling agents of this type areknown in the art and include, but are not limited to, EDCI, HATU, HOBt,DIC and DCC.

As used herein the term “ester” refers to (C₁-C₆) linear or branchedalkyl, (C₄-C₁₀)aryl, (C₄-C₁₀)heteroaryl or arylalkyl esters.

As used herein the term “esterified C-terminus” refers to that theC-terminal of the amino acid in ester form.

As used herein the term “free C-terminus’ refers to that the C-terminalof the amino acid in —CO₂H form.

As used herein, the terms “halogen” or “halo” includes fluorine,chlorine, bromine or iodine.

As used herein the term “Hydroxy” or “Hydroxyl” refers to —OH group.

“Pharmaceutically acceptable salt” is taken to mean an activeingredient, which comprises a compound of the formula (I) in the form ofone of its salts, in particular if this salt form imparts improvedpharmacokinetic properties on the active ingredient compared with thefree form of the active ingredient or any other salt form of the activeingredient used earlier. The pharmaceutically acceptable salt form ofthe active ingredient can also provide this active ingredient for thefirst time with a desired pharmacokinetic property which it did not haveearlier and can even have a positive influence on the pharmacodynamicsof this active ingredient with respect to its therapeutic efficacy inthe body.

“Pharmaceutically acceptable” means that which is useful in preparing apharmaceutical composition that is generally safe, non-toxic, andneither biologically nor otherwise undesirable and includes that whichis acceptable for veterinary as well as human pharmaceutical use.

The term “stereoisomer/stereoisomers” refers to any enantiomers,diastereoisomers, or geometrical isomers of the compounds of formula(I), wherever they are chiral or when they bear one or more double bond.When the compounds of the formula (I) and related formulae are chiral,they can exist in racemic or in optically active form. Since thepharmaceutical activity of the racemates or stereoisomers of thecompounds according to the invention may differ, it may be desirable touse the enantiomers. In these cases, the end product or even theintermediates can be separated into enantiomeric compounds by chemicalor physical measures known to the person skilled in the art or evenemployed as such in the synthesis. In the case of racemic amines,diastereomers are formed from the mixture by reaction with an opticallyactive resolving agent. Examples of suitable resolving agents areoptically active acids such as the R and S forms of tartaric acid,diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malicacid, lactic acid, suitable N-protected amino acids (for exampleN-benzoylproline or N-benzenesulfonylproline), or the various opticallyactive camphorsulfonic acids. Also advantageous is chromatographicenantiomer resolution with the aid of an optically active resolvingagent (for example dinitrobenzoylphenylglycine, cellulose triacetate orother derivatives of carbohydrates or chirally derivatised methacrylatepolymers immobilised on silica gel).

The term “subject” includes mammals (especially humans) and otheranimals, such as domestic animals (e.g., household pets including catsand dogs) and non-domestic animals (such as wildlife).

The phrase “therapeutically effective amount” or “efficient amount”refers to sufficient amount of the compound(s) of the present inventionthat (i) treats or prevents the particular disease, disorder or syndrome(ii) attenuates, ameliorates or eliminates one or more symptoms of theparticular disease, disorder or syndrome or (iii) prevents or delays theonset of one or more symptoms of the particular disease, disorder orsyndrome described herein. In the case of cancer, the therapeuticallyeffective amount of the drug may decrease the number of cancer cells;decrease the cancer size; inhibit (i.e., slow to some extent andalternatively stop) cancer cell infiltration into peripheral organs;suppress (i.e., slow to some extent and alternatively stop) tumormetastasis; inhibit, to some extent, tumor growth; and/or relieve tosome extent one or more of the symptoms associated with the cancer. Inthe case of infectious disease states, the therapeutic effective amountis an amount sufficient to decrease or alleviate an infectious diseases,the symptoms of an infections caused by bacterial, viral and fungal.

Naturally-occurring amino acids are identified throughout thespecification by the conventional three-letter abbreviations indicatedin the below Table 2.

TABLE 2 (Amino acid codes) Name 3-letter code Asparagine Asn Asparticacid Asp Glutamic acid Glu Glutamine Gln Serine Ser Threonine Thr

The abbreviations used in the entire specification may be summarizedherein below with their particular meaning.

° C. (degree Celsius); δ (delta); % (percentage); brine (NaCl solution);CDI CH₂Cl₂/DCM (Dichloromethane); DMF (Dimethyl formamide); DMSO(Dimethyl sulphoxide); DCC (Dicyclohexylcarbodiimide); DIC(N,N′-diisopropylcarbodiimide); DMSO-d₆ (Deuterated DMSO); EDC. HCl/EDCI(1-(3-Dimethyl aminopropyl) carbodiimide hydrochloride); Et₂NH (Diethylamine); EtOH (Ethanol); EtOAc (Ethyl acetate); ECF (ethylchloroformate);Fmoc (Fluorenylmethyloxycarbonyl chloride); g or gr (gram); H or H₂(Hydrogen); H₂O (Water); HOBt/HOBT (1-Hydroxy benzotriazole); HCl(Hydrochloric acid); h or hr (Hours); HATU(2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uranium hexafluorophosphate methanaminium); Hz (Hertz); HPLC (High-performance liquidchromatography); K₂CO₃ (Potassium carbonate); LiOH (Lithium hydroxide);LCMS (Liquid chromatography mass spectroscopy); mmol (Millimoles); M(Molar); μl (Micro litre); mL (Millilitre); mg (Milligram); m(Multiplet); MHz (Megahertz); MS (ES) (Mass spectroscopy-electro spray);min. (Minutes); Na (Sodium); NaHCO₃ (Sodium bicarbonate); NaOAc (Sodiumacetate); NMM (N-methyl morpholine); Na₂SO₄ (Sodium sulphate); N₂(Nitrogen); NMR (Nuclear magnetic resonance spectroscopy); NH₃(Ammonia); NH₂OH.HCl (Hydroxylamine hydrochloride; PD-L1 (Programmeddeath-ligand 1); PD-L2 (Programmed cell death 1 ligand 2);prep-HPLC/preparative HPLC (Preparative High-performance liquidchromatography); S (Singlet); ^(t)Bu (tertiary butyl); TEA/Et₃N(Triethyl amine); TFA (Trifluoroaceticacid); TFAA (Trifluroaceticanhydride); TLC (Thin Layer Chromatography); THF (Tetrahydrofuran); TIPS(Triisopropylsilane); TFA/CF₃COOH (Trifluoroacetic acid); t (Triplet);t_(R)=(Retention time); Trt (Triphenyl methane); etc.

An embodiment of the present invention provides the preparation ofcompounds of formula (I) according to the procedures of the followingexamples, using appropriate materials. Those skilled in the art willunderstand that known variations of the conditions and processes of thefollowing preparative procedures can be used to prepare these compounds.Moreover, by utilizing the procedures described in detail, one ofordinary skill in the art can prepare additional compounds of thepresent invention.

The starting materials are generally available from commercial sourcessuch as Sigma-Aldrich, USA or Germany; Chem-Impex USA; G. L. Biochem,China and Spectrochem, India.

Purification and Characterization of Compounds

Analytical HPLC method: Analytical HPLC was performed using on ZIC HILIC200 A° column (4.6 mm×250 mm, 5 μm), Flow rate: 1.0 mL/min. The elutionconditions used are: Buffer A: 5 mmol ammonium acetate, Buffer B:Acetonitrile, Equilibration of the column with 90% buffer B and elutionby a gradient of 90% to 40% buffer B during 30 min.

Preparative HPLC Method: Preparative HPLC was performed using on SeQuantZIC HILIC 200 A° column (10 mm×250 mm, 5 μm), Flow rate: 5.0 mL/min. Theelution conditions used are: Buffer A: 5 mmol ammonium acetate (adjustto pH-4 with Acetic Acid), Buffer B: Acetonitrile, Equilibration of thecolumn with 90% buffer B and elution by a gradient of 90% to 40% bufferB during 20 min.

LCMS was performed on AP1 2000 LC/MS/MS triple quad (Applied biosystems) with Agilent 1100 series HPLC with G1315 B DAD, using MercuryMS column or using Agilent LC/MSD VL single quad with Agilent 1100series HPLC with G1315 B DAD, using Mercury MS column or using ShimadzuLCMS 2020 single quad with Prominence UFLC system with SPD-20 A DAD.

Example 1 Synthesis of Compound 1

FIG. 1A illustrates Steps 1 a, 1 b and 1 c.

Step 1 a: Ethylchloroformate (1.5 g, 13.78 mmol) and N-Methylmorpholine(1.4 g, 13.78 mmol) were added to a solution of compound 1a (3 g, 11.48mmol) in THF (30 mL) and stirred at −20° C. After 20 min. liquid ammonia(0.77 g, 45.92 mmol) was added to the active mixed anhydride formedin-situ and stirred at 0-5° C. for 20 min. The completeness of thereaction was confirmed by TLC analysis. The reaction mixture wasevaporated under reduced pressure and partitioned between water andethyl acetate. Organic layer was washed with NaHCO₃, citric acid, brinesolution, dried over Na₂SO₄ and evaporated under reduced pressure to get2.9 g of compound 1b (Yield: 96.3%). LCMS: 261.0 (M+H)⁺.

Step 1 b: Trifluroacetic anhydride (9.7 g, 46.0 mmol) was added to asolution of compound 1b (8 g, 30.7 mmol) in pyridine (24.3 g, 307.0mmol) and stirred at room temperature for 3 h. The completeness of thereaction was confirmed by TLC analysis. The reaction mixture wasevaporated under reduced pressure and partitioned between water andethyl acetate. Organic layer was washed with NaHCO₃, citric acid, brinesolution, dried over Na₂SO₄ and evaporated under reduced pressure toafford 7 g of compound 1c (Yield: 94.0%). LCMS: 187.2 (M-^(t)Bu)⁺.

Step 1 c: Hydroxylamine hydrochloride (3 g, 43.37 mmol) and potassiumcarbonate (6 g, 43.37 mmol) were added to a solution of compound 1c (7g, 28.01 mmol) in EtOH (70 mL) and stirred at 90° C. for 2 h. Thecompleteness of the reaction was confirmed by TLC analysis. The reactionmixture was evaporated under reduced pressure and partitioned betweenwater and ethyl acetate. Organic layer was washed with brine solution,dried over Na₂SO₄ and evaporated under reduced pressure. The crudecompound was purified by silica gel column chromatography (Eluent: 0-5%ethyl acetate in hexane) to get 4.2 g of compound 1 d (Yield: 52.8%).LCMS: 276.4 (M+H)⁺.

Step 1 d: Deoxo-Fluor® (1.83 g, 8.3 mmol) was added to a solution ofFmoc-Asn(Trt)-OH (4.5 g, 7.5 mmol) in CH₂Cl₂ (50 mL) and stirred at 0°C. for 3 h. Then CH₂Cl₂ was evaporated and triturated with hexane,decanted and evaporated under vacuum to get the corresponding acidfluoride. NMM (1.17 g, 11.6 mmol) and compound 1d (1.6 g, 5.8 mmol) inTHF were added to the acid fluoride and stirred at room temperature for12 h. Then THF was evaporated and sodium acetate (0.72 g, 8.7 mmol) wasadded followed by EtOH (50 mL). The reaction mixture was stirred at 90°C. for 2 h. The completeness of the reaction was confirmed by TLCanalysis. The reaction mixture was evaporated under reduced pressure andpartitioned between water and ethyl acetate. Organic layer was washedwith NaHCO₃, citric acid, brine solution, dried over Na₂SO₄ andevaporated under reduced pressure, which was further purified by silicagel column chromatography (Eluent: 0-5% ethyl acetate in hexane) toafford 2.8 g of compound 1e (Yield: 44.4%). LCMS: 836.4 (M+H)⁺.

Step 1 e: To compound 1e (2.3 g, 2.7 mmol) in CH₂Cl₂ (10 mL)diethylamine (10 mL) was added and the reaction mixture was stirred atroom temperature for 30 min. The resulting solution was concentrated invacuum to get gummy residue. The crude compound was purified by neutralalumina column chromatography (Eluent: 0-50% ethyl acetate in hexanethen 0-5% methanol in chloroform) to get 1.4 g of 1f (Yield: 90%). LCMS:636.5 (M+Na)⁺.

Step 1 f: To a solution of compound 1f (0.45 g) in CH₂Cl₂ (5 mL),trifluoroacetic acid (5 mL) and catalytic amount of triisopropylsilanewere added and stirred for 3 h at room temperature to remove the acidsensitive protecting groups. The resulting solution was concentrated invacuum to afford 0.29 g of crude compound 1 which was purified usingprep-HPLC method described under experimental conditions. ¹H NMR(DMSO-d₆, 400 MHz): δ 2.58 (m, 2H), 3.53 (m, 3H), 3.91 (t, 1H), 4.36 (t,1H), 6.91 (s, 1H), 7.45 (s, 1H); ¹³C NMR (DMSO-d₆, 400 MHz): δ 20.85,45.71, 50.23, 65.55, 171.03, 171.41, 181.66. LCMS: 216.2 (M+H)⁺; HPLC:t_(R). 13.1 min.

Example 2 Synthesis of Compound 2

FIG. 2 illustrates Steps 2 a and 2b.

Step 2 a: The urea linkage was carried out by the coupling compound 1f(2.7 g, 4.39 mmol) in THF (30 mL) at room temperature with compound 2b(1.67 g, 4.39 mmol). The coupling was initiated by the addition of TEA(0.9 g, 8.78 mmol) in THF (10 m L) and the resultant mixture was stirredat room temperature. After completion of 20 h, THF was evaporated fromthe reaction mass, and partitioned between water and ethyl acetate.Organic layer was washed with water, brine, dried over Na₂SO₄ andevaporated under reduced pressure to get compound 2a, which was furtherpurified by silica gel column chromatography (Eluent: 0-50% ethylacetate in hexane) to afford 3.46 g of compound 2a (Yield: 92.10%). LCMS857.4 (M+H)⁺.

Step 2 b: To a solution of compound 2a (0.22 g, 0.25 mmol) in CH₂Cl₂ (5m L), trifluoroacetic acid (5 mL) and catalytic amount oftriisopropylsilane were added and stirred for 3 h at room temperature.The resulting solution was concentrated under reduced pressure to obtain0.35 g of crude compound. The crude solid material was purified usingpreparative-HPLC method described under experimental conditions. LCMS:347.1 (M+H)⁺; HPLC: t_(R) 12.9 min.

Synthesis of Compound 2b (NO₂—C₆H₄—OCO-Thr (O^(t)Bu)—O^(t)Bu)

To the compound H-Ser(^(t)Bu)—O^(t)Bu (2 g, 9.2 mmol) in CH₂Cl₂ (20 mL),triethylamine (1.39 g, 13.8 mmol) was added and the solution was stirredat room temperature for 5-10 min. To this mixture, solution of4-Nitrophenyl chloroformate (2.22 g, 11.04 mmol) in CH₂Cl₂ was added andthe resultant mixture was stirred at room temperature for 30 min. Thecompletion of the reaction was confirmed by TLC analysis. Aftercompletion of reaction, reaction mixture was diluted with CH₂Cl₂ andwashed with water and 5.0 M citric acid solution, dried over Na₂SO₄ andevaporated under reduced pressure to get crude compound 2b, which wasfurther purified by silica gel column chromatography (Eluent: 0-20%ethyl acetate in hexane) to yield 2.1 g (58.9%) of 2 b.

Example 3 Synthesis of Compound 3

The compound was synthesised using similar procedure as depicted inExample 1 (compound 1) and D-amino acids are linked up in reverse order.Boc-D-Thr(^(t)Bu)—OH was used in place of Boc-Ser(tBu)-OH (compound 1a,Example 1) and Fmoc-D-Asn(trt)-OH in place of Fmoc-Asn(trt)-OH to yield0.15 g crude material of the title compound 3. LCMS: 230.1 (M+H)⁺.

Example 4 Synthesis of Compound 4

The compound was synthesised using similar procedure as depicted inExample 2 for synthesising compound 2 using H-Thr(^(t)Bu)—O^(t)Buinstead of H-Ser(^(t)Bu)—O^(t)Bu (in synthesis of compound 2b) to yield0.35 g crude material of the title compound. The crude solid materialwas purified using preparative HPLC described under experimentalconditions. LCMS: 361.2 (M+H)⁺, HPLC: t_(R)=12.19 min.

Example 5 Synthesis of Compound 5

The compound was synthesised using similar procedure as depicted inExample 4 (compound 4) using D-amino acids are linked up in reverseorder. Boc-D-Thr(^(t)Bu)—OH was used in place of Boc-Ser(^(t)Bu)—OH,Fmoc-D-Asn(trt)-OH in place of Fmoc-Asn(trt)-OH andH-D-Ser(^(t)Bu)—O^(t)Bu was used in place of H-Thr(^(t)Bu)—O^(t)Bu toyield 0.3 g crude material of the title compound. The crude solidmaterial was purified using preparative HPLC described underexperimental conditions. LCMS: 361.3 (M+H)⁺. HPLC: t_(R) 13.58 min.

Example 6 Synthesis of Compound 6

The compound was synthesised using similar procedure as depicted inExample 2 by using H-Thr(^(t)Bu)-OMe instead of H-Ser(^(t)Bu)—O^(t)Bu(in synthesis of compound 2b) to yield 0.2 g crude material of the titlecompound. The crude solid material was purified using preparative HPLCdescribed under experimental conditions. LCMS: 375.1 (M+H)⁺, HPLC:t_(R)=11.84 min.

Example 7 Synthesis of Compound 7

FIG. 3 illustrates Steps 7 a, 7b and 7c.

Step 7 a: The compound 7a was synthesised using similar procedure as forcompound 2a (Example 2, step 2 a) using H-Thr(^(t)Bu)-OMe instead ofH-Ser(^(t)Bu)-OtBu to get crude material which was further purified bysilica gel column chromatography (Eluent: 0-50% ethyl acetate in hexane)to get 2.0 g of compound 7a (Yield: 74%). LCMS: 829.2 (M+H)⁺.

Step 7 b: To a solution of compound 7a (0.35 g, 4.0 mmol) in THF (5 mL)was added lithium hydroxide (0.026 g, 0.63 mmol) at 0° C. and themixture was stirred for 2 h at room temperature. The completion of thereaction was confirmed by TLC analysis. THF was evaporated from thereaction mass, and partitioned between water and ethyl acetate. Organiclayer was washed with citric acid, brine solution, dried over Na₂SO₄ andevaporated under reduced pressure to afford 7b, which was furtherpurified by silica gel column chromatography (Eluent: 0-5% methanol inDCM) to get 0.3 g of product 7b (Yield: 86.7%). LCMS 815.2 (M+H)⁺.

Step 7 c: Compound 7b (0.295 g, 0.39 mmol) was anchored to Rink amideresin (0.7 g, 0.55 mmol/g) using HOBT (0.072 g, 0.54 mmol) and DIC(0.068 g, 0.54 mmol) method in DMF (10 mL). The resin was stirred for 12h at room temperature. The resin was washed with DCM, DMF and DCM anddried. The target compound was cleaved from the rink amide resin usingTFA (5 mL) and catalytic amount of TIPS. The resin was allowed to remainat room temperature for 2 h with occasional stirring. After 2 h, TFA andTIPS were evaporated under nitrogen atmosphere and the resulting residuewas washed with diethyl ether to yield 0.1 g crude material of the titlecompound 7. The crude solid material was purified using preparative HPLCdescribed under experimental conditions. LCMS: 360.0 (M+H)⁺, HPLC:t_(R). 13.88 min.

Example 8 Synthesis of Compound 8

The compound was synthesised using similar procedure as depicted inExample 2 (compound 2) using Fmoc-Glu(O^(t)Bu)—OH instead ofFmoc-Asn(Trt)-OH to get 0.4 g crude material of the title compound. Thecrude solid material was purified using preparative HPLC described underexperimental conditions. LCMS: 362.1 (M+H)⁺. HPLC: t_(R). 13.27 min.

The compounds in Table 3 below were prepared based on the experimentalprocedures described above.

TABLE 3 Compound LCMS HPLC No. Structure (M + H)⁺ (t_(R) in min.) 9

431.1 4.64 10

375.2 11.13 11

361.2 11.85 12

361.2 12.38 13

361.2 12.02 14

375.1 11.74 15

361.1 12.41 16

361.1 12.34 17

361.2 12.62 18

361.2 12.87 19

376.1 12.41 20

375.1 12.31 21

361.3 13.19 22

375.1 12.52 23

389.2 12.07 24

362.2 12.78 25

348.2 13.21

Example 9 Rescue of Mouse Splenocyte Proliferation in Presence ofRecombinant PD-L1/PD-L2

Recombinant mouse PD-L1 (rm-PDL-1, cat no: 1019-B7-100 & rh-PDL-1, catno: 156-B7-100, R&D Systems) were used as the source of PD-L1.

Requirement

Mouse splenocytes harvested from 6-8 weeks old C57 BL6 mice; RPMI 1640(GIBCO, Cat #11875); DMEM with high glucose (GIBCO, Cat #D6429); FetalBovine Serum [Hyclone, Cat #SH30071.03]; Penicillin (10000unit/mL)-Streptomycin(10,000 μg/mL) Liquid (GIBCO, Cat #15140-122); MEMSodium Pyruvate solution 100 mM (100×), Liquid (GIBCO, Cat #11360);Nonessential amino acid (GIBCO, Cat #11140); L-Glutamine (GIBCO, Cat#25030); Anti-CD3 antibody (eBiosciences— 16-0032); Anti-CD28 antibody(eBiosciences— 16-0281); ACK lysis buffer (1 mL) (GIBCO, Cat #-A10492);Histopaque (density-1.083 gm/mL) (SIGMA 10831); Trypan blue solution(SIGMA-T8154); 2 mL Norm Ject Luer Lock syringe-(Sigma 2014-12); 40 μmnylon cell strainer (BD FALCON 35230); Hemacytometer (Bright line-SIGMAZ359629); FACS Buffer (PBS/0.1° A BSA): Phosphate Buffered Saline (PBS)pH 7.2 (HiMedia TS1006) with 0.1% Bovine Serum Albumin (BSA) (SIGMAA7050) and sodium azide (SIGMA 08591); 5 mM stock solution of CFSE: CFSEstock solution was prepared by diluting lyophilized CFSE with 180 μL ofDimethyl sulfoxide (DMSO C₂H₆SO, SIGMA-D-5879) and aliquoted in to tubesfor further use. Working concentrations were titrated from 10 μM to 1μM. (eBioscience-650850-85); 0.05% Trypsin and 0.02% EDTA (SIGMA59417C); 96-well format ELISA plates (Corning CLS3390); BD FACS caliber(E6016); Recombinant mouse B7-H1/PDL1 Fc Chimera, (rm-PD-L1 cat no:1019-B7-100).

Protocol Splenocyte Preparation and Culturing:

Splenocytes harvested in a 50 mL falcon tube by mashing mouse spleen ina 40 μm cell strainer were further treated with 1 mL ACK lysis bufferfor 5 min at room temperature. After washing with 9 mL of RPMI completemedia, cells were re-suspended in 3 mL of 1×PBS in a 15 mL tube. 3 mL ofHistopaque was added carefully to the bottom of the tube withoutdisturbing overlaying splenocyte suspension. After centrifuging at 800×gfor 20 min at room temperature, the opaque layer of splenocytes wascollected carefully without disturbing/mixing the layers. Splenocyteswere washed twice with cold 1×PBS followed by total cell counting usingTrypan Blue exclusion method and used further for cell based assays.

Splenocytes were cultured in RPMI complete media (RPMI+10% fetal bovineserum+1 mM sodium pyruvate+10,000 units/mL penicillin and 10,000 μg/mLstreptomycin) and maintained in a CO₂ incubator with 5% CO₂ at 37° C.

CFSE Proliferation Assay:

CFSE is a dye that passively diffuses into cells and binds tointracellular proteins. 1×10⁶ cells/mL of harvested splenocytes weretreated with 5 μM of CFSE in pre-warmed 1×PBS/0.1% BSA solution for 10min at 37° C. Excess CFSE was quenched using 5 volumes of ice-coldculture media to the cells and incubated on ice for 5 min. CFSE labelledsplenocytes were further given three washes with ice cold complete RPMImedia. CFSE labelled 1×10⁵ splenocytes added to wells containing eitherMDA-MB231 cells (1×10⁵ cells cultured in high glucose DMEM medium) orrecombinant human PDL-1 (100 ng/mL) and test compounds. Splenocytes werestimulated with anti-mouse CD3 and anti-mouse CD28 antibody (1 μg/mLeach), and the culture was further incubated for 72 h at 37° C. with 5%CO₂. Cells were harvested and washed thrice with ice cold FACS bufferand % proliferation was analyzed by flow cytometry with 488 nmexcitation and 521 nm emission filters.

Data Compilation, Processing and Inference:

Percent splenocyte proliferation was analyzed using cell quest FACSprogram and percent rescue of splenocyte proliferation by compound wasestimated after deduction of % background proliferation value andnormalising to % stimulated splenocyte proliferation (positive control)as 100%.

Stimulated splenocytes: Splenocytes+anti-CD3/CD28 stimulationBackground proliferation: Splenocytes+anti-CD3/CD28+PD-L1Compound proliferation: Splenocytes+anti-CD3/CD28+PD-L1+CompoundCompound effect is examined by adding required conc. of compound toanti-CD3/CD28 stimulated splenocytes in presence of ligand (PDL-1)(Table 4).

TABLE 4 Percent rescue of splenocyte Compound proliferation @ 100 nM No.compound concentration 1 93 2 50 4 89 5 67.6 6 84 7 55 8 67 9 34 10 4911 90 12 64 13 74 14 75 15 83 16 72 17 55 18 64 19 88 20 69 21 47 22 5523 74 24 52 25 91

1: A method for treating a cancer in a subject in need thereof,comprising: administering to the subject a compound, wherein thecompound is selected from

or a pharmaceutically acceptable salt thereof; and wherein the cancer isselected from bone cancer, cancer of the head or neck, pancreaticcancer, skin cancer, cutaneous or intraocular malignant melanoma,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, non-Hodgkin's lymphoma, lymphoma,endocrine system, cancer of the thyroid gland, cancer of the parathyroidgland, cancer of the adrenal gland, sarcoma of soft tissue, cancer ofthe urethra, cancer of the penis, leukemia, acute myeloid leukemia,chronic myeloid leukemia, acute lymphoblastic leukemia, chroniclymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma,cancer of the bladder, cancer of the kidney or ureter, carcinoma of therenal pelvis, neoplasm of the central nervous system (CNS), primary CNSlymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma,pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cellcancer, and T-cell lymphoma. 2: The method of claim 1, wherein thecancer is non-Hodgkin's lymphoma. 3: The method of claim 1, wherein thecancer is leukemia. 4: The method of claim 1, wherein the cancer islymphoma. 5: The method of claim 1, wherein the compound is

6: The method of claim 5, wherein the cancer is non-Hodgkin's lymphoma.7: The method of claim 5, wherein the cancer is leukemia. 8: The methodof claim 5, wherein the cancer is lymphoma. 9: The method of claim 1,wherein the compound is a pharmaceutically acceptable salt of

10: The method of claim 9, wherein the cancer is non-Hodgkin's lymphoma.11: The method of claim 9, wherein the cancer is leukemia. 12: Themethod of claim 9, wherein the cancer is lymphoma. 13: The method ofclaim 1, wherein the compound is

14: The method of claim 13, wherein the cancer is non-Hodgkin'slymphoma. 15: The method of claim 13, wherein the cancer is leukemia.16: The method of claim 13, wherein the cancer is lymphoma. 17: Themethod of claim 1, wherein the compound is a pharmaceutically acceptablesalt of

18: The method of claim 17, wherein the cancer is non-Hodgkin'slymphoma. 19: The method of claim 17, wherein the cancer is leukemia.20: The method of claim 17, wherein the cancer is lymphoma.